Cold‐active microbial enzymes and their biotechnological applications

Abstract Microorganisms known as psychrophiles/psychrotrophs, which survive in cold climates, constitute majority of the biosphere on Earth. Their capability to produce cold‐active enzymes along with other distinguishing characteristics allows them to survive in the cold environments. Due to the relative ease of large‐scale production compared to enzymes from plants and animals, commercial uses of microbial enzyme are alluring. The ocean depths, polar, and alpine regions, which make up over 85% of the planet, are inhabited to cold ecosystems. Microbes living in these regions are important for their metabolic contribution to the ecosphere as well as for their enzymes, which may have potential industrial applications. Cold‐adapted microorganisms are a possible source of cold‐active enzymes that have high catalytic efficacy at low and moderate temperatures at which homologous mesophilic enzymes are not active. Cold‐active enzymes can be used in a variety of biotechnological processes, including food processing, additives in the detergent and food industries, textile industry, waste‐water treatment, biopulping, environmental bioremediation in cold climates, biotransformation, and molecular biology applications with great potential for energy savings. Genetically manipulated strains that are suitable for producing a particular cold‐active enzyme would be crucial in a variety of industrial and biotechnological applications. The potential advantage of cold‐adapted enzymes will probably lead to a greater annual market than for thermo‐stable enzymes in the near future. This review includes latest updates on various microbial source of cold‐active enzymes and their biotechnological applications.


INTRODUCTION
Most of the ecosystems on the Earth are subjected to temperatures that are consistently below 5°C.The ecosphere's cold ecosystems, which include seas, soils, glaciers, lakes, and sea ice, make up more than 70% of the total area (Feller & Gerday, 2003).The vast majority of Earth's biosphere is made up of microbes called psychrophiles and psychrotrophs that can survive in extremely cold conditions.True psychrophiles are adapted not only to low temperatures but also, sometimes, to additional environmental limitations.They are subjected to extraordinarily high pressures in the ocean depths and sediments; hence, they must be called as piezo-psychrophiles as well (Yayanos, 1995).Microorganisms that have adapted to the cold are a possible source of heat-sensitive, homologous cold-active enzymes that have high catalytic effectiveness at low and mild temperatures.The potentials of cold-active enzymes and their producing organisms have been periodically evaluated, and they have a wide range of current and future applications in biotechnology (Kuddus, 2015).These enzymes are highly catalytic at low and moderate temperatures where homologous mesophilic enzymes are inactive.In biotechnology, this characteristic shortens low-temperature process durations, saves energy, prevents chemical transformations, and minimizes volatile molecular loss.Cold-active enzymes support low-temperature processes that preserve heat-labile compounds and maintain product quality.It allows selective enzyme inactivation in complicated media without expensive heating/cooling equipment (Margesin, 2009).
Scientific and industrial groups have put in a lot of work over the past few decades to find new cold-active enzymes with desirable features for usage in various biotechnological procedures (Mangiagalli & Lotti, 2021).Molecular biology, detergents, food and drink processing can all benefit from the cold-adapted enzymes because of their increased activity at lower temperatures.In addition, the selective deactivation of enzymes in complicated combinations is possible through the use of thermo-sensible enzymes.The ability to perform biotechnological activities more efficiently, economically with sustainability than with hightemperature-adapted enzymes is what makes them so appealing and in demand (Barroca et al., 2017).The food, beverages, biofuels, and detergent sectors all make use of distinct techniques that call for psychrozymes such as amylases, cellulases, invertases, proteases, and lipases.In addition to its use in lactose-free dairy products, coldactive galactosidases have found a home in the culinary, cosmetic, and pharmaceutical industries.These enzymes are distinguished by their optimum catalytic activity at temperatures between those considered low and adequate; nevertheless, they are also heat-sensitive and rapidly inactivated over a certain temperature (Hamid et al., 2022).Due to their high efficiency and environmentally friendly nature, enzymes are in great demand in many industrial applications, including food and beverage production (Grzonka et al., 2007;Moharram et al., 2022;Schäfer et al., 2006;Singh et al., 2016).Moreover, these enzymes are an essential component of a well-established global sector that is projected to increase in value over the next several years to US$6.3 billion (Dewan, 2017;Moharram et al., 2022;Singh et al., 2016).These days, it's common practice to use cold-active enzymes to bring down the temperature of industrial processes.This reduces energy consumption and carbon emissions, and it also increases productivity because these processes work better at ambient or lower temperatures (Białkowska & Turkiewicz, 2014;Pulicherla et al., 2011;Sarmiento et al., 2015).Cold-active enzymes are employed in biotechnology to limit the loss of volatile components and stop a variety of undesirable reactions (Gerday, 2013;Kuddus, 2018;Kumari et al., 2021;Santiago et al., 2016).It is anticipated that the application of cold-active enzymes would significantly grow during the coming decades (Al-Ghanayem & Joseph, 2020;Santiago et al., 2016).Cold-active enzymes with different biotechnological applications and examples are shown in Figure 1.

HABITATS OF COLD -ADAPTED MICROORGANISMS AND THEIR COLD -ACTI VE ENZ YMES
All the cold-adapted microorganisms grouped into two broad categories viz.psychrophiles, which thrive at temperatures below 15°C and psychrotrophs, which can survive at temperatures between 20 and 25°C (Farrell & Rose, 1967;Moyer & Morita, 2007).The name "psychrophile" is of Greek origin ("Psychros" means cold and "Philos" means loving), i.e. cold-loving (Schmidt-Nielsen, 1902).Psychrophiles are usually found in deep | 3 of 19 COLD-ACTIVE MICROBIAL ENZYMES oceans, icebergs, and frozen areas of land such as glaciers and snow-covered areas; and also isolated from cold-water lakes, cold deserts, and caves where the temperature is below 10°C (Hamid et al., 2022).About 85% of Earth is covered by various cold habitats, both manmade and natural, and within these environments, a wide variety of cold-adapted microorganisms including archaea, bacteria, fungi, yeasts, and viruses are thriving (Margesin & Collins, 2019).Almost three-quarters of all known forms of life on the Earth are psychrophiles, which survive in extreme environments such as Antarctica, high-altitude soils, glaciers, deep sea waters (both fresh and marine), and the mountains (Hamid et al., 2022).
Most of the cold-active enzymes are isolated from cold-adapted microorganisms; however, some enzymes active at low temperatures were isolated from mesophilic microorganisms (Bhatia et al., 2021;Hamid et al., 2022;Santiago et al., 2016).These enzymes have high catalytic efficiency and protein flexibility at low temperature along with low thermal stability due to their unique protein characteristics (Rojas-Contreras et al., 2015).On the other hand, some of the cold-active enzymes showed high thermal stability that have an added advantage for various industrial applications (Zhou et al., 2019).Microorganisms such as bacteria, fungi, and algae were found to be excellent sources of cold-active enzymes and their sources have been reviewed (Bhatia et al., 2021;Hamid et al., 2022;Santiago et al., 2016).A list of cold-active enzymes recently isolated from various cold-adapted microorganisms and their habitat is summarized in Table 1.Five psychrophilic Gram-negative bacteria were isolated from maritime sediments of Svalbard by Knoblauch et al. (1999).Sánchez et al. (2009) isolated thirty bacterial colonies from South Atlantic Argentina.A promising source of antibacterial activity has been discovered in the isolated bacterium, closely related to Serratia sp.(Sánchez et al., 2009).Metabolically active psychrophiles capable of reproducing at −15°C (Planococcus halocryophilus Or1) were isolated from Arctic permafrost (Mykytczuk et al., 2013).The above-mentioned microorganisms are considered unmined treasures for industrial and biotechnological applications.Unculturable cold-adapted microbes that have various potentials in producing metabolites are explored through the metagenomics approach (Vester et al., 2015).This helps to increase knowledge and gives us a better understanding of novel enzymes that can be used in biotechnological applications.

COLD -ACTI VE ENZ YMES IN FOOD PROCESSING/FOOD INDUSTRY
For the food scientists and biotechnologists, the current issue is the discovery of novel enzymes for their commercial applications.Cold-active enzymes are excellent biocatalysts with strong specific activity at low temperature that do not require heating processes, which impede the quality, sustainability, and costeffectiveness of industrial production.The cold-shock proteins of psychrophilic bacteria, which include enzymes like pectinase, proteases, amylases, lipases, and cellulases, have numerous biotechnological applications in the food-processing sector (Gounot, 1991).Cold-active galactosidase with an optimal temperature range of 15-18°C has opened a new field of research in the dairy and food processing industry (Hamid et al., 2022).Some of the important cold-active enzymes used in the food industry are described below and summarized in Figure 2.

Lactase
Lactose, a type of carbohydrate, makes up the bulk of our daily diet.β-galactosidase, more generally known as lactase, is an enzyme responsible for hydrolysing lactose into glucose and galactose (Shukla & Wierzbicki, 1975).The food processing industry is using β-galactosidase at large scale.Cold-adapted β-galactosidase is a significant food enzyme, which removes lactose from the milk at low temperatures or in refrigerators so that it can be consumed by lactoseintolerant peoples.Also, the cheese industry's waste product, whey, is converted to more easily fermentable glucose and galactose using cold-adapted βgalactosidase.Recent studies have characterized a marine psychrophilic bacterium, which produces coldactive β-galactosidase that digest over 80% of the lactose in raw milk at 20°C and pH 6.5, indicating the possibility of using them in the dairy sector at industrial scale (Ghosh et al., 2012;Pulicherla et al., 2013).Potential cold-active β-galactosidases could be used to produce low-cost lactose-free products.

Amylase
Amylase is one of the most essential industrial enzymes used in food, textile, paper, and fermentation industries.In biotechnology, these enzymes are especially used for the hydrolysis of starch.Cold-active amylases present novel possibilities for biotechnological exploitation due to their strong catalytic activity at low temperature, low thermostability, and peculiar specificities.Alteromonas haloplanktis, an Antarctic bacterium, was the first microorganism producing cold-adapted αamylase, which was extensively studied and has been magnificently expressed in the mesophilic host E. coli (Feller et al., 1998).Kuddus et al., isolated cold-active α-amylase producing Microbacterium foliorum from Gangotri glacier, Western Himalaya, India with potential biotechnological applications (Kuddus et al., 2012).The structure and function of cold-active α-amylases T A B L E 1 Some important cold-active enzymes and their microbial source.and their biotechnological potentials are reviewed by Kuddus et al. (2011).

Pectinase
The pectinase or pectinolytic enzymes are crucial components of the food processing industry.Pectinolytic enzymes, which are utilized in the food, paper, textile, and juice industries, make up around 10% of the enzyme market (Garg et al., 2016;Kashyap et al., 2001;Semenova et al., 2006)

T A B L E 1 (Continued)
F I G U R E 2 Cold-active enzymes used in different sectors of food industry.Pulicherla et al., 2011).Because grape juice and other fruit juices must have a pH of between 2.5 and 3.5, researchers have been searching for pectinases that can function at both low temperatures and low pH (Adapa et al., 2014).Many studies have been done on the isolation and characterization of cold-active pectinases from microorganisms.Recently, three new species producing cold-active pectinases viz Cladosporium parasphaerospermum, Cladosporium chlamydosporigenum, and Cladosporium compactisporum are reported (Moharram et al., 2022).The purified cold-active pectinase enhanced the yield of apple, orange, apricot, and peach juice and improved the clarity and colour of orange juice (Moharram et al., 2022).Another study reported pectinases from Antarctic Geomyces sp.F09-T3-2 that may be potentially suitable for biotechnological applications needing cold-active pectinases (Poveda et al., 2018).
Pectinases are also used to improve the cloud stability of fruit nectars, clarify fruit juices and wines, and process coffee and tea (Reid & Ricard, 2002;Soares, 2001).

Lipase
For the esterification of fats and the generation of fatty acids, cold-active lipases are used (Jaeger and Eggert, 2002).The lipases produced by psychrophilic bacteria are a crucial enzyme in today's food processing industries and become an integral part of the industry in many ways.Various cold-active lipase-producing microorganisms and their source are already reported (Kavitha, 2016).Cold-active lipases have been utilized to change flavours and enhance the consistency of many foods.To generate antioxidants for use in sunflower oil, functionalized phenols were esterified using cold-active lipases from C. antarctica, Candida cylindracea AY30, Hansinuela lanuginosa, Pseudomonas sp., and G. candidum (Buisman et al., 1998;Pandey et al., 1999).Cold-active lipase from Pseudomonas strain P38 is commonly employed in nonaqueous biotransformation for butyl caprylate n-heptane production (Tan et al., 1996).

Protease
The most important use of cold-active proteases, which are produced by psychrophilic microorganisms, is in developing the flavour and enhancing the tenderness of meats stored in the refrigerator.In the food industry, cold-active proteases are useful due to their ability to maintain product integrity at low temperatures.Rapid inactivation of thermally labile enzymes, such as coldactive proteases, can be achieved by subjecting them to mild heat (Margesin et al., 2002) which is beneficial in the process management.Protein hydrolysates that are both highly digestible and nutritionally beneficial can be prepared by using cold-active alkaline proteases at low temperature.Protein hydrolysate is beneficial in blood pressure management and as an ingredient in infant food formulas and also used to fortify fruit juices and soft beverages for medicinal purposes (Neklyudov et al., 2000).Other possible applications of cold-active proteases in the food industry are reviewed by Yang et al. (2023).

COLD -ACTI VE ENZ YMES IN DETERGENT INDUSTRY
Household or industrial dirt and stains mainly in the form of proteins, lipids, and starch components are difficult to remove from the fabrics.Most of these stains are removed by heating and beating which shortens the life of fabrics (Rahman et al., 2023).Enzyme-based detergents play an important role in the removal of dirt/stains.However, in the cold regions it is difficult to remove the dirt/stains by using detergents that contain mesophilic enzymes.The addition of chemicals to replace the enzymes increases environmental pollution and is harmful.Also, mesophilic enzymes with narrow physiochemical activities become inactive under low temperatures.In such conditions, cold-active enzymes are gaining more attention due to their low-temperature activity and thermostability (Hamid et al., 2022).Enzymes active under low temperatures with alkaline stability help to retain the quality of fabrics and fulfil consumer needs.Further cold-active enzymes improve washing performance with low energy requirements and preserve the quality and life of fabrics.These enzymes are biodegradable, which reduce pollution and are safe for aquatic organisms.Washing machine manufacturers are targeting machines to perform cold washing and thereby reducing energy consumption and protecting the fabrics (Sarmiento et al., 2015).Enzymes such as lipase, protease, and amylase are normally used as detergent additives.Lipases hydrolyse lipids and thereby it removes the lipid stains.Cold-active lipases are capable of removing the lipid stain even at low temperatures.Proteases are enzymes that hydrolyse peptide bonds.The protein stains such as egg stains, bloodstains, grass, etc. are removed by the addition of cold-active proteases in the detergent.Several cold-adapted microorganisms isolated from different cold regions have been reported for cold-active protease production (Kuddus & Ahmad, 2012;Kuddus & Ramteke, 2011).Some stains, such as sauces, vegetable stains, gravy, cereals, etc., are starch-based stains and are removed easily by using amylases as a detergent additive (Niyonzima & More, 2014).Amylases break the bonds between glucose units in the linear amylose chain of starch by cleaving glycosidic linkages.Amylases contribute around 25% of total enzyme sales and are used in 90% of the detergent formulation (Hmidet et al., 2009).Some studies on detergent-resistant pullulanase, which hydrolyse the α-1,6 glucosidic linkages, are also reported (Wu et al., 2023).Along with coldactive protease and lipase, cellulase is also used as a detergent additive that helps in removing stains by cleaving cellulose polymers.It increases the brightness of the fabric, along with reducing the fuzz and pills, especially in woollen garments.Cellulose is also used for increasing the finishing and smoothness of fabrics (Karmakar & Ray, 2011).In cotton fabrics, dirt and damaged fibres are accumulated in between cotton fibres.The dirt present in inter-fibrils is also removed or digested by enzymatic action (Niyonzima, 2019).Mannanase and pectin stains are also difficult to remove from fabrics.Cold-active mannanase and pectinase have a great importance to be an additive in detergents to improve the efficiency of detergent formulations (Kim et al., 2008).However, till date coldactive pectinases and mannanase for its compatibility with detergents are not well explored.

COLD -ACTI VE ENZ YMES IN TE X TILE INDUSTRY
In the textile industries, enzymes including cellulases, catalases, and laccases are frequently used.These enzymes are capable of destroying starch, degrading excessive hydrogen peroxide, decomposing materials, and tainting lignin.Cold-active enzymes are increasingly being used in the textile industry due to their extraordinarily clear, productive, non-toxic, and environment friendly qualities.Some of the cold-active enzymes generally used in the textile industry include cellulases and proteases.
Cellulases catalyse the hydrolysis of cellulose into shorter oligosaccharides and ultimately in glucose.In the textile industry, cold-active cellulase plays a critical role economically in the biopolishing of fabric and stone-washing of jeans at low temperatures (Gerday et al., 2000).It is also used to remove fine fluff on the surface of cotton fabric under low-temperature conditions (Bhat et al., 2013).Proteases are used in the textile industry to increase the softness of raw silk by removing its stiff and unappealing gum coating of sericin.The surface of wool and silk fibres can be altered by protease treatments to provide novel and distinctive finishes (Benmrad et al., 2018).Textile companies can make advantage of cold-active proteases (Singhal et al., 2012).When every step is carried out at low temperature, cloth can be protected from the damaging effects of high temperatures for an extended period.Because synthetic fibres cannot withstand temperatures beyond 50-60°C, cold-active proteases provide us with a novel and unique method of clearance of new fabrics (Guduk et al., 2019;Joshi & Satyanarayana, 2013).

COLD -ACTI VE ENZ YMES IN WASTE-WATER TRE ATMENT
Wastewater contains different types of chemicals, oils, polysaccharides, and other contaminants that reduce the oxygen content, which leads to the death of aquatic organisms (Bashir et al., 2020).A high content of these materials even affects the treatment of wastewater.Wastewater discharge from households, restaurants, and food industries in cold regions needs more attention due to the formation of clogs and flocs (Mahto & Das, 2022).Emulsified oil in the wastewater of cold regions has density variation and is more viscous with lesser interfacial films to stabilize the oil phase.To separate the flocs needs high-cost treatment, in this context use of cold-active enzymes in pretreatment reduces the issues at low temperatures.Wastewater treatment by using cold-active enzymes is expensive due to the costly process of enzyme production.An alternative method such as inoculating cold-adapted enzyme-producing bacteria or cell immobilization or enzyme immobilization reduces the costs of treating the wastewater at low temperature.
Cold-active lipases play an important role in reducing oil contamination in wastewater (Hassan et al., 2018).Lipases act on the interface between the substrate and aqueous phase and different types of lipases with unique substrate specificity are essential to dissolve lipid contaminants in the wastewater (Nimkande & Bafana, 2022).Another major component of wastewater is starch molecules that are hydrolysed into smaller glucose units with the help of alpha-amylase enzymes.Chemical degradation of starch molecules is now substituted by microbial amylase due to its substrate specificity, stability, and easy manipulation.Cold-active amylase with pH and substrate stability can be used in the treatment of wastewater in cold regions.Another major concern is proteinaceous waste from food and household effluents that interferes with BOD in the aquatic system.These materials can be treated with crude enzymes or direct microbial inoculation of coldadapted proteolytic bacteria for reducing the production cost of pure cold-active enzymes (Furhan, 2020).Wastewater from textile, animal feed and paper industries contains cellulose and related components.Cold-active cellulase plays an important role in hydrolysing cellulose materials in wastewater.Biosurfactants are also used in the treatment of wastewater, that reduce the surface tension between the air-water interface and bioemulsifiers that reduce the interfacial tension between immiscible liquids or solid materials.These biosurfactants and bioemulsifiers increase the availability of substrates to the enzymes even in cold climatic conditions.Cold-active enzymes can be used in the treatment of waste materials from candy, confectionary, leather, paper, meat and wine industries in cold regions (Duarte et al., 2018).Cold-active pectinase (polygalacturonase) was produced from Geotrichum sp by using waste from the fruit and vegetable industries (Divya & Padma, 2015).Cold-tolerant chitinases were produced from Antarctic fungus Lecanicillium muscarium CCFEE-5003 by using shrimp and crab wastes, so it may be used in the treatment of chitin-containing wastewater from shrimp and crab industries (Barghini et al., 2013).Apart from these applications, coldactive enzymes are also reported for treating harmful chemicals such as petroleum hydrocarbons, alkanes, aromatic hydrocarbon, chlorinated hydrocarbons, polycyclic aromatic hydrocarbons, and petroleum hydrocarbons in wastewater in cold regions (Park & Park, 2018).

COLD -ACTI VE ENZ YMES IN BIOPULPING
Biopulping is an environment friendly process that employs biological methods in lieu of chemical agents to separate lignin from cellulose fibres within wood chips, with the aim of reducing use of chemicals and enhancing sustainability (Worku et al., 2023).It involves the microbial pretreatment of wood chips with the concept centred on the colonization of wood by microbes that selectively degrade lignin while largely preserving cellulose (Ferraz et al., 2008;Mendonça et al., 2008).Conventional pulping methods heavily rely on the utilization of toxic chemicals like sodium hydroxide (NaOH) and sodium sulfide (Na 2 S), which can result in adverse environmental consequences.In contrast, biopulping predominantly uses fungi, enzymes, and biological methods to break down lignin, promoting an environmentally friendly approach (Worku et al., 2023).However, in the context of biopulping, there are instances where controlled and limited chemical usage may persist.This could involve the application of enzymes or other biological agents that have been derived from chemical processes.Moreover, chemicals may occasionally play a role as facilitators in the biopulping process to enhance efficiency or accelerate reactions (Kumar et al., 2020).Biotreatment can yield several benefits, including reduced pulping time, lower consumption of bleaching chemicals, improved pulp strength, enhanced delignification rates, decreased alkali usage, and energy savings during defibration and refining phases (Islam et al., 2008;Mendonça et al., 2008).These advancements offer solutions to certain challenges in traditional chemical and mechanical pulping methods.Combining biopulping with mechanical pulping results in a sustainable approach that significantly increases mill throughput and reduces electrical energy consumption while producing stronger pulp with longer fibres and increased fibrillation.The introduction of specific lignin-degrading microorganisms can rapidly modify wood cell walls (Bajpai, 2018).
Biopulping has the potential to revolutionize the paper and pulp industry by offering a more sustainable alternative to traditional pulping methods.The use of biological methods instead of chemicals can reduce the environmental impact of the pulping process and potentially lead to improved product quality (Bhardwaj et al., 2019).While biopulping is currently in its early stages of development, it holds significant promise as a more sustainable alternative to conventional pulping methods.The biopulping process with microbial cold enzymes typically entails introducing these enzymes to the wood chips, allowing them to react over a specified period.Subsequently, the fibres undergo a washing step to eliminate any remaining enzymes, preparing them for use in papermaking (Azeez, 2018).Biopulping can be more time-consuming and expensive compared to traditional pulping methods.Therefore, it is crucial to strike the right balance between sustainability and cost-effectiveness (Kumar et al., 2020).Cold biopulping employs enzymes like pectinases, hemicellulases, and cellulases to break down pectin, hemicellulose, and cellulose, respectively.The utilization of cold enzymes in biopulping has demonstrated the potential to reduce pulping time, increase pulp yield, and potentially enhance the brightness and strength of the final product (Hamid et al., 2022).One of the primary advantages of using cold-active enzymes in biopulping is their capacity to reduce the energy requirements of the pulping process, making it a more sustainable and eco-friendly choice.Additionally, these cold-active enzymes including cellulases and hemicellulases can enhance the yield and quality of the final pulp product.For example, cellulases and hemicellulases break down cellulose and hemicellulose into smaller sugars, which can be used as an energy source or as raw materials for other processes, leading to improved paper strength and brightness (Wei et al., 2021).
While biopulping holds immense potential, there are several challenges that must be addressed before it can be widely adopted in the industry.Key challenges include scaling up the process for commercial production, improving the efficiency and speed of the pulping process, and reducing associated costs (Kumar et al., 2020).Despite these challenges, some companies have already begun to integrate biopulping into their operations.Startups and research institutions, for instance, are exploring the use of fungi and enzymes to break down lignin in wood chips (Goodell et al., 2020).Established companies in the paper and pulp industry are also investing in biopulping research and development with the aim of bringing this technology to the market.Additionally, the utilization of cold enzymes in biopulping remains a dynamic area of research, with ongoing efforts to optimize the enzymes and their application conditions to maximize effectiveness and efficiency (Wei et al., 2021).Microbial cold-active enzymes are often used in biopulping because they are effective at breaking down lignin at low temperatures, which results in less energy consumption and a lower carbon footprint compared to traditional pulping methods (Joseph et al., 2008).In addition, microbial enzymes are biodegradable and non-toxic, making them a more sustainable alternative to chemical pulping methods (Kumar & Rani, 2019).
Enzymatic biobleaching is a highly effective technique that supports the breakdown of hemicellulose and aids in the removal of lignin.Microbial xylanases are utilized in biobleaching to enhance the extractability of lignin in subsequent phases without directly damaging it.These xylanases target the lignin-carbohydrate complex, leading to the removal and modification of xylan and glucomannan structures, resulting in improved delignification efficiency (Çiçekler & Tutuş, 2019;Colonia et al., 2019;Zakaria et al., 2015).Xylanases, among various enzymes, exhibit great potential in the pulp and paper industries.They interact with xylan in wood, breaking down lignin-carbohydrate linkages, thereby enhancing paper quality when applied in biopulping and biobleaching (Gupta et al., 2013;Pandey et al., 2022).Cold-active xylanases play a crucial role in various low-temperature processes, including biopulping, biobleaching, and applications in clarifying fruit juices and textiles.Their high catalytic activity at lower temperatures makes them excellent biocatalysts (Collins et al., 2005;Suhaib et al., 2018).These enzymes find efficient use in diverse industrial and biotechnological processes, spanning molecular biology, food and beverages, detergent production, medical applications, paper manufacturing, and textiles (Hamid, 2021;Wackett, 2019).
Xylanases are particularly valuable in reducing the need for oxidizing chemicals by approximately 20%, resulting in improved brightness during biobleaching processes.This is attributed to their bleach-boosting properties (Bajaj & Mahajan, 2019;Garg et al., 2011;Shrinivas et al., 2010).One limitation of enzyme utilization in biopulping is their limited penetration into wood chips, making their effectiveness somewhat challenging.To address this, enzymes have historically been applied to partially defibrated raw wood material or during the bleaching of chemical pulp (Burton, 2001;Eugenio et al., 2010;Lehr et al., 2021).Alternatively, the use of vacuum or pressure can expedite the migration of enzymes into wood chips (Maijala et al., 2008).Another essential enzyme in the paper industry is endoβ-mannanase.This enzyme hydrolyses linear glucomannan, mannan, and 1,4-glycosidic linkages within the mannan chain in a random manner.The resulting hydrolysate, composed of mannan-oligosaccharides, must undergo conversion to monosaccharides through the action of the enzyme mannosidase (Hlalukana et al., 2021).Enzyme-treated fibres may exhibit a slight reduction in inter-fibre bonding strength when cellulose is present, but this does not compromise the mechanical strength of the fibres (Valls et al., 2010).In the absence of cellulose, xylanase application leads to increased viscosity, while hemicellulose hydrolysis eases lignin removal (Li et al., 2010;Walia et al., 2017).
The application of psychrozymes in biopulping represents a promising advancement within the paper and pulp industry.These microbial cold enzymes have the potential to play a significant role in the industry's future by reducing the energy requirements for pulping and providing a more environmentally friendly alternative to traditional pulping methods (Wei et al., 2021).Cold enzymes play a vital role in the biopulping process by breaking down lignocellulosic materials in wood and agricultural waste into smaller components.Cold enzymes enable the breakdown of lignin, cellulose, and hemicellulose at lower temperatures compared to conventional pulping processes, which typically involve high temperatures and chemical treatments (Kumar & Rani, 2019).However, the utilization of cold enzymes in biopulping may pose some challenges, such as the requirement for high enzyme concentrations and extended reaction times.Additionally, the cost associated with producing and purifying cold enzymes can limit their widespread adoption in industry.It is crucial to note that while biopulping aims to reduce the use of chemicals in the pulping process, there may still be situations where chemicals are used, but their application should be minimal and rigorously controlled to ensure that the environmental benefits of biopulping are not compromised.The use of chemicals should be transparent, and their environmental impact should be thoroughly assessed (Kumar et al., 2020).Biopulping has the potential to play a pivotal role in the future of the paper and pulp industry.Despite the challenges that need to be addressed, the potential advantages offered by cold-active enzymes in biopulping make it a promising avenue for ongoing research and development.The mesophilic enzymes used in biopulping such as xylanase for xylan degradation (Walia et al., 2017), laccase for lignin degradation (Zerva et al., 2019), cellulase for cellulose degradation (Li et al., 2019), and pectinase for pectin degradation (Haile & Ayele, 2022) may be replaced by cold-active xylanase, laccase, cellulase, and pectinase, respectively; that improve pulp and paper quality and also decreases energy demands.Research efforts are continuing to optimize cold enzymes and their application in biopulping, making them an attractive option for the future of the pulp and paper industry (Mesbah, 2022).

COLD -ACTI VE ENZ YMES FOR ENVIRONMENTAL BIOREMEDI ATION IN COLD CLIMATES
Environment is contaminated by various activities including industrial, tourist activities, human migration, transportation etc. that become a serious threat to human populations and create serious environmental hazards.Effective solution for this problem is bioremediation due to less destruction and economic benefits.Enzymes are highly efficient green biocatalysts with huge energy savings, reducing chemical reactions, non-toxic, biodegradable, safe, cost-efficient, and ecofriendly (Bilal et al., 2019).Psychrophilic microorganisms and its coldactive enzymes are gaining attention due to its activity under harsh conditions and suitability in industrial and biotechnological application with low activation energy.Majority of the Earth's portion experiences cold climates ranging from Antarctica to Arctic, high mountains to deep sea, lakes etc., that are subjected to contamination (Rota et al., 2022).Microorganisms in these regions play an important role in nutrient recycling, mineralization and biodegradation with the help of enzymes adapted to such extreme conditions.Pollution in cold regions is increasing tremendously with petroleum hydrocarbons, chlorinated chemicals, solvents, pesticides, etc.These pollutants remain much longer in cold regions because of low bioavailability, lack of microbial populations adapted to low temperature and harsh climatic conditions.Several factors also affect the process such as properties of the pollutant, metabolic limitations, mass transfer through cell membrane, change in temperature, bioavailability, oxygen, electron acceptors, toxicity, and freeze-thaw process (Yang et al., 2009).In cold regions, the biodegradation is affected by low temperature, affecting the physical nature that reduces the bioavailability of the microbial community.Further, the increased viscosity and decreased volatility affect the biodegradation (Bajaj & Singh, 2015).The higher specificity and catalytic activity at low temperature makes cold-active enzymes appropriate for bioremediation (Kumar et al., 2021).Bioremediation in cold regions, contaminated with certain pollutants, are carried out by bioaugmentation methods.There are certain limitations for these strategies including low nutrient availability, poor availability of pollutants to the microbial populations and efficiency of the strain to degrade particular chemicals.To overcome these limitations a thorough understanding of bioremediation of pollutants in cold regions is needed.Several studies were conducted to implement bioremediation strategies in the cold regions by using psychrophilic microbial consortia, cold-active enzymes and recombinant bioaugmentation (Chaudhary & Kim, 2019;Davoodi et al., 2020).
Many of the cold-active enzymes producing bacteria and fungi were reported for the bioremediation process.Due to the increasing pollution and climatic variations, the number of microbes is decreasing in temperate regions.Inoculations of consortia-producing cold-active enzymes helped in boosting the degradation of hydrocarbons (Gerday et al., 2000).However, for minimizing the environmental contamination and for bioremediation in cold regions a huge economical input is required to achieve rapid and successful results.The degree of success in bioremediation in cold regions is affected during seasonal transition.Cold-adapted microorganisms in appropriate populations that are tolerant to harsh environmental conditions perform biodegradation at in-situ conditions for the removal of pollutants (Miri et al., 2022).The efficient biodegradation with cold-active enzymes acquired by structural adaptation of proteins through genetic modifications and long-term selection process (Struvay & Feller, 2012).The variation in amino acid compared to mesophilic enzymes makes weaker interaction in protein making it flexible at low temperature (Feller, 2010).Lowering the activation energy because of weak substrate binding subsequently increases the rate of the reaction.Thus psychrophilic microorganisms and cold-active enzymes play a major role in bioremediation of pollutants in cold regions compared to mesophilic counterparts.
Bioremediation in Arctic and sub-Arctic regions has been achieved successfully, but removal of pollutants from the cold regions faces many challenges compared to other regions.To preserve the environment there is a need to reconsider, adapt and improve the current strategies in cold environments.Therefore, In Situ methods such as bioaugmentation and biostimulation are adopted.Bioaugmentation in cold regions is the addition of cold-adapted microorganisms capable of degrading a particular pollutant to speed up the rate of biodegradation (Yuan et al., 2018).Studies were conducted to determine the effect of bioaugmentation in polluted areas of cold regions (Filler et al., 2008;Hosokawa et al., 2011).In cold regions, bioaugmentation becomes successful when the native population capable of degrading pollutants is low (Camenzuli & Freidman, 2015;Patel et al., 2018).Biostimulation is another process by which nutrients are supplied to soil to stimulate the native microbial population.The presence of native degrading microorganisms enriched bioremediation in cold regions contaminated with hydrocarbons (Simpanen et al., 2016).A pre-optimized biostimulation process removed 75% of the pollutants in 40 days (Martínez Álvarez et al., 2017).Another method of bioremediation is addition of free water that enhances the microbial load in soil.Only a few reports are available on this method due to the lesser efficiency compared to the above-mentioned two methods (Stallwood et al., 2005).The increased proportion of microbial load in bioaugmentation and bio-stimulation degrades the pollutant effectively (Kuhn et al., 2009).

COLD -ACTI VE ENZ YMES IN BIOTR ANSFORMATION AND MOLECUL AR BIOLOGY APPLICATIONS
Cold-active enzymes play a pivotal role in biotransformation, particularly in frigid environments such as the Arctic and Antarctic regions (Peng et al., 2022).These enzymes exhibit functionality at low temperatures, where conventional enzymes would remain inactive, rendering them valuable tools in biocatalysis and bioprocessing at low temperatures.Moreover, the utilization of cold-active enzymes in biotransformation offers several advantages, including decreased energy consumption, reduced production costs, and enhanced reaction efficiency (Jin et al., 2019).Furthermore, the integration of cold-active enzymes into biotransformation processes contributes to a reduction in environmental impacts by curbing waste generation and diminishing the overall carbon footprint of the operation.Consequently, the study and application of coldactive enzymes in biotransformation have emerged as a critical area of research with the potential to revolutionize diverse industries, such as food processing, pharmaceuticals, and biofuels (Carrasco et al., 2012;Gupta et al., 2020).Cold-active lipases find applications in bioremediation, biotransformation, baking, food processing, as well as in molecular biology and the detergent sector (Joseph et al., 2008).In summary, the role of cold-active enzymes in biotransformation holds promise for sustainable and efficient bioprocessing and biocatalysis, particularly in frigid environments.For instance, cold-active chitinase enzymes and certain yeast psychrozymes are employed in the conversion of chitin into bioethanol (Dahiya et al., 2006).Future research endeavours in this domain may encompass the exploration and optimization of novel cold-active enzymes, alongside the development of innovative biotransformation processes that fully harness the unique attributes of these enzymes.
Cold-active enzymes are characterized by their ability to remain active at low temperatures while being vulnerable to heat, making them valuable assets in various fields including molecular biology (Cavicchioli et al., 2011;Kuddus, 2018;Marx et al., 2007;Sahay et al., 2013).Microbial enzymes possess a range of distinctive properties, such as stability, eco-friendliness, high productivity, flexibility, and economic viability, among others.These attributes have amplified the importance of microbial enzymes across multiple industries (Gurung et al., 2013;Mangiagalli & Lotti, 2021).Various researchers have reported on the application and potential of cold-active enzymes in molecular biology and food biotechnology (Hamid, 2021;Hamid et al., 2022;Kuddus, 2018).The heat sensitivity associated with cold-adapted enzymes is a critical feature for sequential enzymatic processes used in various molecular biology techniques (Kumar et al., 2021).In molecular biology, alkaline phosphatases (AP) are widely used, including in cloning, where they are employed to dephosphorylate the 5′ end of a linearized DNA fragment, preventing its recircularization (Sharma et al., 2014).Traditionally, the only available APs were derived from E. coli or calf intestinal tissue and required heat-resistant deactivation with detergents, which could potentially interfere with subsequent processes.In contrast, psychrophilic heat-labile APs can be quickly inactivated in the same tube by subjecting them to a mild heat treatment at 65°C (Hamdan, 2018).The first heat-labile AP was discovered in a bacterium from Antarctica and was purified in 1984.Commercially produced as Antarctic phosphatase by New England Biolabs, it was initially isolated from another Antarctic bacterium and further improved through directed evolution (Koutsioulis et al., 2008).In 1993, ArcticZymes in Tromsø, Norway, introduced the first commercially available cold-adapted AP, using the Arctic shrimp Pandalus borealis.In 2010, ArcticZymes unveiled a genetically engineered version of this AP.Recent metagenomics library creation from ocean-tidal flat sediments on the west coast of Korea led to the discovery of a novel psychrophilic AP (Lee et al., 2015) with properties and effectiveness similar to other commercially available APs. Numerous approaches and techniques have been developed for in-depth studies of the structural and molecular aspects of cold-active lipases (Do et al., 2013;Jeon et al., 2009;Parra et al., 2008).
In molecular biology, cold-adapted enzymes find further applications: (a) Uracil-DNA N-glycosylase (UNG) catalyses the release of free uracil from uracil-con (Schormann et al., 2014).UNG is employed in various applications, including preventing carry-over contamination in PCR and RT PCR, site-directed mutagenesis, and SNP genotyping (Tetzner, 2009).ArcticZymes offers a commercially available cold-adapted UNG enzyme derived from Atlantic cod (Gadus morhua) through recombinant DNA technology.This enzyme is entirely and irreversibly inactivated by heat treatment at 55°C (Lanes et al., 2002).Another heat-labile UNG was discovered in the genome of the bacterium Psychrobacter sp.HJ147, and it was cloned and expressed using E. coli.It functions optimally between 20 and 25°C and has a half-life of two minutes at 40°C (Lee et al., 2009).New England Biolabs provides an Antarctic Thermolabile UDG, a recombinant UNG enzyme created in E. coli from a psychrophilic marine bacterium, which remains active at temperatures above 50°C.(b) Double-strandspecific DNase can digest double-stranded DNA without affecting single-stranded DNA molecules like primers or probes.This enzyme is used for genomic DNA extraction from RNA preparations or PCR cleanup (Rittié & Perbal, 2008).ArcticZymes offers a heat-labile version of this enzyme, originally isolated from shrimp (Pandalus borealis) and subsequently genetically modified to become inactive at 55°C.Affymetrix USB (Santa Clara, CA, USA) provides a recombinant version produced in Pichia pastoris, which is inactivated after exposure to 70°C for 25-30 min.(c) Cryonase, a recombinant cold-active nuclease from a psychrophilic strain of Shewanella sp, is available from Takara-Clontech (Mountain View, CA, USA).This enzyme, created in E. coli, can digest any form of DNA or RNA, including single-stranded, double-stranded, linear, or circularized molecules (Bruno et al., 2019).Cryonase remains active even when samples are kept on ice and can be completely inactivated with a 30-min incubation at 70°C.
Nucleases play a crucial role in breaking down DNA or RNA and are essential for removing contaminating nucleic acids from the reaction mixture (Yang, 2011).However, after their activity, it is necessary to eliminate the nucleases from the reaction mixture.Typically, this is achieved by heating the enzyme mixture to deactivate it.In such cases, the use of cold-adapted nucleases offers advantages as they can be rapidly and effectively inactivated by mild heat treatments.Cold-adapted nucleases sourced from psychrophilic microorganisms, including Pandalus borealis and Shewanella sp., are commercially available (Nandanwar et al., 2020).Furthermore, the exploration of a cold-adapted nuclease derived from a psychrophilic strain of Psychromonas ingrahamii for potential applications in molecular biology is promising (Maciejewska et al., 2019).Another study identified a cold-adapted RNase enzyme from Psychrobacter, a bacterium found in Antarctic Sea ice, opening up new avenues for research and study in molecular biology (Wang et al., 2019).Ligases, on the other hand, facilitate the formation of phosphodiester links between two DNA fragments, allowing the joining of these DNA fragments.Bacteriophages are commonly used as a source of ligases for this purpose.In molecular biology, the optimal temperature for the activity of DNA ligases is often low, making cold-adapted ligases advantageous (Nandanwar et al., 2020).Recent research has described three cold-adapted DNA ligases from psychrophilic species, highlighting their optimal temperatures and thermal stabilities.Further studies may explore their applications in molecular biology (Berg et al., 2019).

COLD -ACTI VE ENZ YMES: SUSTAINABLE SOURCE FOR BIO -BASED ECONOMY
Cold-active enzymes play a crucial role in the biobased economy by enabling biotechnological processes to occur at low temperatures.This is significant because many industrial processes traditionally require high temperatures, which consume more energy and may not be suitable for temperature-sensitive materials or organisms.Cold-active enzymes offer several advantages in various sectors of the bio-based economy.
Cold-active enzymes function optimally at low to moderate temperatures (often below 50°C), reducing the energy required for heating in industrial processes.This contributes to the overall sustainability of bio-based production systems with energy efficiency.It involves the preservation of heat-sensitive compounds.In industries like food, pharmaceuticals, and cosmetics, cold-active enzymes can be used to process and manufacture products without compromising the integrity of heat-sensitive compounds (Kuddus et al., 2011).This is crucial for preserving the quality and functionality of these products.As for environmental impact, lowering the temperature requirements for industrial processes reduces greenhouse gas emissions, as less energy is needed for heating.This aligns with the goals of a more sustainable and environmentally friendly bio-based economy.Cold-active enzymes are employed in environmental bioremediation processes also.They can help clean up cold environments, such as polar regions, where traditional enzymes might be less effective due to low temperatures.Cold-active enzymes are also used in cold food processing where it can be used to improve processes like cheese production, beer brewing, and yogurt making, all of which require low-temperature fermentation or maturation.In biofuel production, enzymes used in the production of biofuels from lignocellulosic biomass often work more efficiently at lower temperatures.Cold-active enzymes can play a role in breaking down lignocellulose into fermentable sugars for bioethanol or biogas production.It can play a significant role in biorefineries for the efficient conversion of biomass into biofuels and other valuable chemicals.
Cold-active enzymes also have significant impact in medical and pharmaceutical applications.These enzymes are valuable in biopharmaceutical production processes, including the expression and purification of temperature-sensitive proteins and the production of vaccines and biologics.Cold-active enzymes along with its producing organisms can be applied in wastewater treatment systems in regions with colder climates, helping to degrade pollutants and organic matter more efficiently.In addition, it used in aquaculture, such as those in fish feed, may benefit from coldactive enzymes as they allow for optimal digestion in the lower-temperature environments of fish tanks or open water systems.Cold-active enzymes are of interest to researchers and innovators in biotechnology and synthetic biology for their unique properties.Scientists can explore their potential in novel applications and develop new biotechnological processes.In summary, cold-active enzymes are essential components of the bio-based economy, contributing to increased sustainability, reduced energy consumption, and the expansion of biotechnological applications in various sectors.Their unique ability to function at low temperatures opens up new possibilities for efficient, environmentally friendly processes and products.

CONCLUSION AND FUTURE PROSPECTS OF COLD -ACTI VE ENZ YMES
The scope of cold-active enzymes is promising, as they continue to gain importance in various industries and applications.Their ability to function at lower temperatures can enhance the efficiency of enzymatic hydrolysis and fermentation processes.In pharmaceutical and biopharmaceutical production processes, they can aid in the expression, purification, and formulation of temperature-sensitive proteins and biologics, improving yields and product quality.Cold-active enzymes will continue to be utilized in the food and beverage industry to improve the quality and processing of products like dairy and beverages.In regions with cold climates, cold-active enzymes can be employed in environmental cleanup efforts, such as the remediation of oil spills and other pollutants in cold-water ecosystems.It can contribute to more efficient feed conversion in aquaculture, leading to improved sustainability and reduced environmental impact.In agriculture, these enzymes may be applied to enhance nutrient availability and soil health in colder climates.As research into extremophiles advances, cold-active enzymes from these organisms could be used in various industrial applications, including those requiring low temperatures.Advances in enzyme engineering techniques could lead to the creation of tailor-made cold-active enzymes with enhanced properties, stability, and specificity for specific applications.Cold-active enzymes might find applications in medical research and diagnostics, potentially assisting in the development of novel diagnostic assays and therapies that require low-temperature conditions.It could play a role in enhancing the efficiency of cold-chain logistics, ensuring the preservation and quality of temperaturesensitive products during storage and transportation.Ongoing research into the molecular mechanisms and properties of cold-active enzymes could uncover new insights that drive innovation in various fields, potentially leading to breakthroughs in areas like enzyme stability, activity, and substrate specificity.Advances in bioinformatics and metagenomics could lead to the discovery of novel cold-active enzymes from unexplored environments, further expanding the enzyme toolbox for various applications.Overall, the future prospects of cold-active enzymes are closely tied to the growing demand for sustainable, energy-efficient, and environmentally friendly solutions across industries.

C O N F L I C T O F I N T E R E S T S TAT E M E N T
There is no conflict of interest.

Cold-active enzyme Cold-adapted microorganism Habitat of microorganism Temp. of habitat Reference
Abbreviation: NM, not mentioned.